could also replenish long-lived ASCs throughout life. Virus-specific ASCs tran-

siently circulate in the peripheral tissues at the acute phase of an infection and

survive in the bone marrow from months to years.

The outline of various steps involved in the production of human antibodies using

single-cell PCR is summarized in Fig. 22.3. In brief, it involves isolation of periph-

eral blood mononuclear cells (PBMCs) from naturally infected or vaccinated human

subjects. The PBMCs are sorted by flow cytometer into ASCs (IgG⁺IgD
; CD19⁺

CD3
CD20^low and then sub-gated as CD27^high CD38^high) and memory B cells

(CD27⁺) into 96-well PCR plates. In humans actively immunized with influenza

vaccine, ASCs peaked around day 7, whereas the peak of memory B cells has been

observed from days 14 to 21 after vaccination (Wrammert et al. 2008). VH and Vκ

genes from each cell are amplified in a one-step RT-PCR reaction using a cocktail of

sense primers specific for the leader regions and antisense primers to the Cγ constant

regions for heavy chains and Cκ constant region for the light chain. Subsequently,

Fig. 22.3 Outline of the procedure to generate fully human antibody using single-B cell PCR:

Peripheral blood mononuclear cells (PBMCs) are isolated from the blood of either naturally infected

or immunized human subjects, followed by purification of single antibody-secreting cells (ASCs)/

memory B cells by flow cytometer into 96-well PCR plates. Subsequently, light and heavy chains of

the antibody from single cells are amplified by RT-PCR using sense primers specific for the leader

region of heavy and light chains and antisense primers to the Cγ constant regions for heavy chains

and Cκ for the light chain. The amplified light and heavy chain fragments are cloned into VH and

VL expression vectors. The mammalian cells are co-transfected with the VH and VL expression

vectors. Single-cell clones are isolated by using appropriate drugs as selection markers and

antibody-secreting clones are identified by high-throughput screening assays, followed by antibody

expression, purification, and characterization

22

Therapeutic Human Monoclonal Antibodies

411